Anti-angiogenic approaches to treat tumors have-shown great promise in experimental animal models. However, to date it is unclear whether such therapeutic approaches will be successful in man. Efforts to improve anti-angiogenic tumor therapy have focused on vascular targeting strategies. Two of the project leaders, Drs. Schnitzer and Ruoshlati, in this program have been pioneers in the identification of vascular targets. One such target is the integrin alpha-v-beta3, which is expressed on angiogenic but not on normal blood vessels. Accordingly, we developed a non-viral gene delivery strategy that selectively targets integrin alpha-v-beta3 on angiogenic tumor-associated blood vessels in mice. Delivery of a mutant form of Raf-1 kinase to these blood vessels caused extensive apoptosis of tumor vessels which lead to the subsequent apoptosis and necrosis of the tumor itself. In this proposal we will extend our studies on targeted gene delivery to the tumor vascular in three new directions. First, as a means to test the physiological relevance of this approach, we will extend this targeted gene delivery to establish its capacity to influence the growth and metastasis of a spontaneous tumor in mice. We also will chemically conjugate the F3 peptide (Ruoslahti) and Annexin 1 antibody (Schnitzer) to a small molecule Raf inhibitor for targeted delivery to the tumor vascular. This Raf inhibitor given systemically has shown some efficacy in cancer patients. We will test the hypothesis that this agent, when targeted to tumor vessels, will produce a more robust anti-tumor response. Second, in conjunction with Dr. Dieseroff, we will test the hypothesis chemotherapeutic treatment of the tumor can synergize with our approach to target genes to the tumor vasculature. In addition, we will evaluate the role that Raf-1 plays in endothelial cell and tumor cell survival by delivery of point mutants of Raf that suppress two separate sites within its activation domain. Finally, we will also evaluate the mechanism by which alpha-v-beta3-targeted nanoparticles become endocytosed and utilize additional targeted ligands identified by Drs. Ruoshlati and Schnitzer as a means to target new genes to the tumor vasculature. These studies should provide a basis to proceed toward the clinical development of a targeted gene delivery approach to suppress the growth of tumor-associated blood vessels.